POS0808 MICA/B-DEPENDENT ACTIVATION OF CYTOTOXIC NATURAL KILLER CELLS BY INFLAMMATORY CDC2 CONTRIBUTE TO PRIMARY SJÖGREN´S SYNDROME PATHOLOGY
Authors: I. Sánchez-Cerrillo, M. Calvet-Mirabent, A. Triguero-Martinez, D. Calzada Fraile, C. Delgado-Arévalo, M. Valdivia, M. Ramirez, E. Vazquez de Luis, A. Benguría-Filippini, R. Moreno, M. Adrados de Llano, H. De la Fuente, I. Tsukalov, E. Roy Vallejo, A. Ramino, S. Iborra, F. Sánchez-Madrid, A. Dopazo, I. González-Álvaro, S. Castañeda, E. Martin-Gayo
Journal: Annals of the Rheumatic Diseases (2023)

<h3>Background</h3> Primary Sjögren´s syndrome (pSS) is an example of an inflammatory autoimmune disorder largely mediated by IFN responses, leading to damage of exocrine glands that has been linked to autoreactive adaptive immune cells, such as Th17, CD8+ T cell and B cell [1, 2, 3]. However, the potential contribution of different innate immune cells such as Natural Killer (NK) cells and dendritic cells (DC) to pSS pathology remains understudied. <h3>Objectives</h3> We identify the molecular mechanisms regulating pathogenic crosstalk between NK and DC in pSS using samples from pSS patients and an experimental <i>in vivo</i> SS model. <h3>Methods</h3> Phenotypical analysis of myeloid and NK cell subsets in PBMCs from 47 pSS patients and 56 healthy donors (HD) was performed by flow cytometry. Histological analysis of SG from n=7 pSS was performed by confocal microscopy. Cytotoxic function of NK cells was assessed by culture with a K562-GFP target cell line. Transcriptional analysis of sorted Mo, CD1c+ and CD141+ cDC from four pSS patients and four HD was performed by RNA-seq. Co-culture between sorted NK cells, Mo and CD1c+ or CD141+ cDCs was used for test functional interactions. Regulation of ligands for NK cell receptors on cDC was analyzed by FACS after stimulation with poly I:C in the absence or the presence of specific siRNAs. Finally, <i>a</i>ltered DC and NK cell phenotypes and interactions with Th17 and B cells were analyzed in a murine Sjögren-mouse model induced by poly I:C intraperitoneal injections [4] in which we used a depleting anti-NK1.1 antibody or an isotype control each 4 days. <h3>Results</h3> Here, we identified an enriched transitional CD16<sup>+</sup> CD56<sup>hi</sup> NK cell subset in pSS individuals associated with higher NK cell cytotoxic function <i>in vitro</i>. In addition, elevated proportions of inflammatory CD64<sup>+</sup> cDC2 exhibiting increased levels of MICa/b (p= &lt;0,01), the ligand for the activating receptor NKG2D, were observed in the blood of these patients. Circulating cDC2 from pSS were capable of efficiently inducing activation of cytotoxic NK cells <i>ex vivo</i> and were found near CD56<sup>+</sup> NK cells in salivary glands (SG) from pSS patients. Interestingly, cDC2 from pSS were characterized by preferential transcriptional activation of IFN signatures associated to the RIG-I/DDX60 pathway and its target genes. These sensors regulate the expression of MICa/b ligands on cDC2. Finally, increased proportions of CD64<sup>hi</sup> cDC2 (p=&lt;0,0001) expressing RAE-1 (p= &lt;0,01), a murine activating NKG2D ligand, and transitional NKG2D<sup>+</sup> CD11b<sup>+</sup> CD27<sup>+</sup> NK cells (p=&lt;0,001) were present <i>in vivo</i> in the SG of an <i>in vivo</i> model of pSS. Remarkably, depletion of NK cells during the inflammation onset prevented subsequent induction of IL-17<sup>+</sup> CD4 (p= &lt;0,01) and memory IgD<sup>-</sup> IgM<sup>-</sup> CD38<sup>hi</sup> B cells (p=&lt;0,0001) in the SG. <h3>Conclusion</h3> Thus, our study provides novel innate immune cellular and molecular mechanisms contributing to pSS pathology and identifies new potential therapy targets. <h3>References</h3> [1]Lin X, Rui K, Deng J, Tian J, Wang X, Wang S, et al. Th17 cells play a critical role in the development of experimental Sjögren's syndrome. Annals of the rheumatic diseases. 2015;74(6):1302-10. [2]Hansen A, Odendahl M, Reiter K, Jacobi AM, Feist E, Scholze J, et al. Diminished peripheral blood memory B cells and accumulation of memory B cells in the salivary glands of patients with Sjögren's syndrome. Arthritis and rheumatism. 2002;46(8):2160-71. [3]Emamian ES, Leon JM, Lessard CJ, Grandits M, et al. Peripheral blood gene expression profiling in Sjögren's syndrome. Genes Immun. 2009;10(4):285-96. [4]Nandula SR, Scindia YM, Dey P, et al. Activation of innate immunity accelerates sialoadenitis in a mouse model for Sjögren's syndrome-like disease. Oral diseases. 2011;17(8):801-7. <h3>Acknowledgements:</h3> NIL. <h3>Disclosure of Interests</h3> Ildefonso Sánchez-Cerrillo: None declared, Marta Calvet-Mirabent: None declared, Ana Triguero-Martinez: None declared, Diego Calzada Fraile: None declared, Cristina Delgado-Arévalo: None declared, Mariel Valdivia: None declared, Marta Ramirez: None declared, Enrique Vazquez de Luis: None declared, Alberto Benguría-Filippini: None declared, Roberto Moreno: None declared, María Adrados de LLano: None declared, Hortensia de la Fuente: None declared, Ilya Tsukalov: None declared, Emilia Roy Vallejo: None declared, Almudena Ramino: None declared, Salvador Iborra: None declared, Francisco Sánchez-Madrid: None declared, Ana Dopazo: None declared, Isidoro González-Álvaro Consultant of: Lilly and Sanofi, Santos Castañeda: None declared, Enrique Martin-Gayo Grant/research support from: Gilead.